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Publication Abstract from PubMed

Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a K(i) of 52 nM. The zinc- and iron-containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pK(a) of 6.0, and Zn-CDA has a kinetic pK(a) of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on k(cat) and k(cat)/K(m), consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed.

Three-dimensional structure and catalytic mechanism of Cytosine deaminase.,Hall RS, Fedorov AA, Xu C, Fedorov EV, Almo SC, Raushel FM Biochemistry. 2011 Jun 7;50(22):5077-85. Epub 2011 May 12. PMID:21545144^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.